Vanadium haloperoxidase enzymes are useful in industrial catalysis in a variety of contexts (Sheffield, et al., Biotechnology Techniques, 8:579–582 (1994)). For instance, they catalyze a variety of halogenation, oxidation and epoxidation reactions (Itoh, et al., Eur. J. Biochem., 172:477 (1988); Itoh, et. al. Biochimica et Biophysica Acta., 1994 (1993); Itoh, et al., Appl. Microbiol. & Biotechnol., 43:394–401 (1995)). Although a halide ion is a required cofactor for enzyme activity, products may not be halogenated. Numerous uses in synthetic organic chemistry include reactions with diverse substrates such as aliphatic and aromatic hydrocarbons, phenols, β-diketones and nitrogen- and sulfur-containing heterocycles (Itoh, et al., Eur. J. Biochem. 172:477 (1988); Neidleman et al., Biohalogenation: Principles, Basic Roles and Applications, Ellis Horwood, John Wiley & Sons, New York (1986)). Bromoperoxidases can also be used in place of synthetic organic chemistry reactions to make activated intermediates or products such as pesticides. In addition, these enzymes have an advantage over chemical synthesis in producing stereospecific products (Itoh, et al., Eur. J. Biochem., 172:477 (1988)). Moreover, haloperoxidases have unusual stability (both temporal and thermal) and are active in solvents including methanol, ethanol and acetone.
Recent medical applications of bromoperoxidase have been described. Lovqvist, et al., Nuclear Medicine and Biology, 22:125–131 (1995) described the enzymatic bromination of a monoclonal antibody with BR-radionuclide for imaging of antibody localization by PET scanning. There is current interest in enzymatic production of antibiotics including fosfomycin and pyrrolnitrin (Itoh, et. al. Biochimica et Biophysica Acta. 1994 (1993); Itoh, et al., Appl. Microbiol. &Biotechnol. 43:394–401 (1995)) and 7-chlorotetracycline (van Pée, K. H., J. Bacteriol., 170:5890–5894 (1988)) via haloperoxidase-catalyzed reactions in bacteria.
Known haloperoxidases include bromoperoxidases from brown and red algae including Fucus and Ascophyllum (Butler, et al., Chem. Rev., 93:1937–1994 (1993)), iodoperoxidase from green algae (Mehrtens, G., Polar Biol. 14:351–354 (1994)), and chloroperoxidase from the fungus Curvularia inaequalis (Van Schijndel, et al., Eur. J. Biochem., 225:151–157 (1994)). A vanadate requirement for algal haloperoxidase was first described by Vilter (Vilter, H., Biological Systems, 31, Vanadium and its role in life, Sigel, et al. (Eds.), Marcel Dekker, New York, N.Y., pp. 325–362 (1995)).
The specific bromoperoxidase activity of the native Fucus enzyme is several fold higher (Butler, et al.) than the other algal enzymes for which at least partial sequences have been reported, Ascophyllum (Vilter 1995) and Corallina (Shimonishi, et al. FEBS Letters, 428, 105–110 (1998)), and higher specific activity than the Curvularia fungal chloroperoxidase (van Schijndel et al. BBA 1161:249–256 (1993)).
Extracted and partially purified bromoperoxidase from the red alga Corallina officinalis is commercially available from Sigma Chemical Company. Sigma has also investigated immobilization of enzyme on agarose beads (Sheffield, et al., Phytochemistry, 38:1103–1107 (1995)) and on cellulose acetate membrane (Sheffield, et al., Biotechnology Techniques, 8:579–582 (1994)) for repetitive catalysis of bromination reactions in flow-through reactors in enzyme-driven preparative organic chemistry. Many industrial uses for stable soybean peroxidase are envisioned by A. Pokora of Enzymol International, Inc. as described by Wick (Wick, C. B., Genetic Engineering News, 16(3):1, 18–19). Recombinant enzyme biotechnology is of current industrial interest because enzymes are safe, low-polluting alternatives to chemicals in many applications, and can be modified by protein engineering to fit the requirements of specific applications (Kelly, E. B. Genetic Engineering News, 16(5):1, 30, 32 (1996) Lovqvist, et al., Nuclear Medicine and Biology, 22:125–131 (1995)). Peroxidases can also be incorporated into moldable plastics (Service, R. F., Science 272:196–197 (1996)).
Multiple representatives of other classes of peroxidases have been produced in recombinant form. A heme peroxidase, manganese peroxidase from the fungus Phanerochaete chrysosporidium, was expressed in recombinant form and refolded for activity (Whitwam, R. E., Biochem. Biophys. Research. Communications, 216:1013–1017 (1995)). Recombinant horseradish peroxidase isozyme C (a heme peroxidase) for use in chemiluminescent labeling in molecular biology and biotechnology applications has been described (EP 0299682, WO 89/03424 ). Recombinant non-heme haloperoxidases have been prepared from the bacteria, Pseudomonas pyrrocinia (Wolfframm, et al., Gene 130:131–135 (1993)) and two related Streptomyces aureofaciens enzymes (van Pée, K. H., J. Bacteriol., 170:5890–5894 (1988); Pfeifer, et al., J. Gen. Microbiol. 138:1123–1131 (1992)).
The X-ray structure for the fungal chloroperoxidase from Curvularia has been reported Messerschmidt and Wever., Proc. Natl. Acad. Sci. U.S.A. 93:392–396 (1996). The chloroperoxidase consisted of two helical bundle domains organized as an N-terminal noncatalytic bundle and a C-terminal catalytic bundle. The X-ray structure of the bromoperoxidase from the brown alga Ascophyllum (Weyand et al J. Mol. Biol. 293:595–611 (1999)) and the bromoperoxidase from the red alga Corallina (Littlechild Curr, Opin. Chem. Biol. 3:28–34 (1999)) have been published.
Despite the interest in vanadium haloperoxidases, there are relatively few reports in the literature of the cloning and recombinant expression of a vanadium haloperoxidases. Shimonshi et al. FEBS Lett. 428:105–110 (1998) described cloning of the enzyme from Corallina pilulifera. Cloning of the Curvularia gene is described by Hemrika, et al. PNAS 94,2145–2149 (1997) and 95/27046. A partial sequence of the Ascophyllum gene is described in Vilter (1995). There is a need in the art for efficient means for producing vanadium haloperoxidases using techniques such as recombinant expression. The present invention addresses these and other needs.